The design and fabrication of precise spatial patterns, microstructures, and nanostructures of peptides and proteins have widespread applications in tissue engineering, cellular biology, molecular electronics, biosensors, photonics, delivery of biomolecules, and therapeutics. Micro and nanostructured scaffolds and cellular substrates in two and three dimensions can directly influence the spatial organization of tissues and organs. Engineered structures of peptides, proteins, and DNA have applications in the delivery of bioactive molecules and therapeutics. The spatial arrangement of biomolecules in a controlled manner on a surface can enable fundamental biochemical analyses of complex systems, screening, and multiplexed assays. Periodic structures have the ability to manipulate the passage of light resulting in novel metamaterials and photonic crystals. To achieve these structures, diverse techniques including self-assembly, soft lithography, and photolithography have been reported.
Known approaches to spatially arrange proteins have relied on passive, substrate-based methods in which chemical modifications to the substrate induce preferential regions for adhesion. In three dimensions, the ability to spatially pattern proteins can enable the formation of intricate scaffolds for the growth of cells in scaffolds. To date, this has been restricted to simplistic architectures achieved using methods such as casting alone. Such methods do not provide adequate micro and nanoscale control.
Photolithography (“writing using light”) using ultraviolet to visible light has been widely used in the semiconductor industry to pattern materials and fabricate circuits of extraordinary complexity with microstructured spatial morphologies. However, a primary requirement for such light activated processes is that the material they act upon must be light sensitive, i.e. photocurable. This has limited the scope of application of photolithography to a narrow base of starting materials, typically synthetic polymers. One approach to directly pattern proteins has been to alter them chemically or biochemically to render them intrinsically photoreactive. Examples include photactivable derivatives of biotin, and elastin-like proteins with photo-reactive, non-canonical amino acids incorporated via site-specific and residue-specific techniques. To date, neither of the strategies described have been shown with intact naturally available biopolymers. Significantly, light-based systems that can handle different compositions of proteins are not efficiently developed. Thus, the development of a modified photolithography platform is attractive as a means of directly producing spatially-immobilized biomolecules in 2D and 3D.
Silks have a rich history as a biomaterial either by themselves or blended/complexed with polymers, ceramics and gold. Silkworm silk, in particular, is a unique biocompatible material that has used in textile processing and medical applications for thousands of years. Silk exists in a self-assembled fibrous configuration, in which mechanically robust fibroin (70%) comprises the core of the silk fiber, and a glue protein sericin (30%) surrounds this core. Silk is classified as an FDA approved material and defined by the USP as a nondegradable material because it retains over 50% of its tensile strength 60 days post-implantation in viva.
Silk fibroin, in contrast to naturally occurring silk comprising both the proteins fibroin and sericin, is the more commonly used biomaterial. It is slowly absorbed in vivo and degrades over time based on several different factors, with typical proteolytic degradation and resorption within a year. In contrast to synthetic materials, the degradable behavior of silk fibroins does not result in an adverse immunogenic response. This is significant advantage over materials that have the potential to create adverse reactions either by themselves or due to their degradation products in vivo. Developing new processing strategies to enhance fibroin applications therefore has great biomedical potential.
Furuzono et al. (Chemical modification of silk fibroin with 2-methacryloyloxyethyl phosphorylcholine. II. Graft-polymerization onto fabric through 2-methacryloyloxyethyl isocyanate and interaction between fabric and platelets. Biomaterials, 2000. 21(4): p. 327-333) describes work on isocyanate addition to a silk protein, using 2-Methacryloyloxyethyl isocyanate/isocyanatoethyl methacrylate (MOI/ICEMA). This group acted as a stepping stone for adding a further functionality onto silk ‘fabric’ or degummed fiber. Furuzono did not solubilize the silk protein and substantially lost the isocyanate functionality upon further reaction.
Teramoto et al. (Chemical Modification of Silk Sericin in Lithium Chloride/Dimethyl Sulfoxide Solvent with 4-Cyanophenyl Isocyanate. Biomacromolecules 2004, 5, (4), 1392-1398) took advantage of similar chemistry a few years after Furuzono's work. Teramoto et al. used a solvent system of 1M LiCl/DMSO, to achieve isocyanate addition on solubilized sericin but did not contemplate or address making sericin photoreactive.